(Actinopterygii) in the State of Rio De Janeiro, Brazil

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NEMATODE AND CESTODE LARVAE OF HYGIENIC-SANITARY IMPORTANCE IN Lopholatilus villarii (ACTINOPTERYGII) IN THE STATE OF RIO DE JANEIRO, BRAZIL

Aline Monteiro da SILVA1,2, Marcelo KNOFF2*, Nilza Nunes FELIZARDO1, Delir Corrêa GOMES2, Sérgio Carmona de SÃO CLEMENTE1

ABSTRACT The tile fish, Lopholatilus villarii, occurs from Brazil to Argentina where it is commercially important it because possesses highly appreciated meat. The aim of this study was to identify the larvae of anisakid and raphidascaridid nematodes and trypanorhynch cestodes in L. villarii purchased in fish markets of the municipality of Niterói, state of Rio de Janeiro, Brazil,and calculate their parasitological indices and present their sites of infection. From August 2015 to September 2016, 31 specimens of L. villarii were investigated. The fish were necropsied and their viscera and musculature analyzed. The helminths found were processed according to standard helminthological techniques for taxonomic identification. Of 31 fish analyzed, 28 (90.3%) were parasitized with larvae of anisakid and raphidascaridid nematodes, Anisakis sp., Terranova sp.,

Pseudoterranova sp. and Hysterothylacium deardorffoverstreetorum (L3 and L4) and plerocercus of the trypanorhynch cestode Otobothrium sp. Their parasitological indices were calculated. The helminths parasitized mainly the serosas of intestine, stomach and liver, and H. deardorffoverstreetorum was also parasitizing the abdominal musculature of one fish. This is the first report of anisakid and raphidascaridid nematodes and trypanorhynch cestodes in L. villarii. Hygienic-sanitary aspects were discussed. Key words: Malacanthidae; Anisakidae; Raphidascarididae; Trypanorhyncha.

LARVAS DE NEMATOIDES E CESTOIDES DE IMPORTÂNCIA HIGIÊNICO-SANITÁRIA EM Lopholatilus villarii (ACTINOPTERYGII) NO ESTADO DO RIO DE JANEIRO, BRASIL

RESUMO O peixe batata, Lopholatilus villarii, ocorre do Brasil a Argentina e possui uma carne apreciada e comercialmente importante. O objetivo deste estudo foi identificar as larvas de nematoides Anisakidae e Raphidascarididae e cestoides Trypanorhyncha de L. villarii obtidos em mercados de pescado do município de Niterói, Estado do Rio de Janeiro, Brasil, calcular seus índices parasitários e apresentar os sítios de infecção. De agosto de 2015 a setembro de 2016, foram investigados 31 espécimes de L. villarii. Os peixes foram necropsiados e suas vísceras e musculatura e analisados. Os helmintos encontrados foram processados de acordo com as técnicas em helmintologia para permitir a identificação taxonômica. Dos 31 peixes analisados, 28 (90,3%) estavam parasitados com larvas de nematoides anisaquídeos e rafidascaridídeos: Anisakis sp., Terranova sp., Pseudoterranova

sp. e Hysterothylacium deardorffoverstreetorum (L3 e L4) e plerocercos de cestoides Trypanorhyncha: Otobothrium sp. Seus índices parasitários foram calculados. Os helmintos estavam parasitando, principalmente, as serosas do intestino, estômago e fígado e H. deardorffoverstreetorum estava também na musculatura abdominal de um peixe. Este é o primeiro registro de nematoides anisaquídeos e rafidascaridídeos e cestoides Trypanorhyncha em L. villarii. Aspectos higiênico- sanitários foram discutidos. Palavras-chave: Malacanthidae; Anisakidae; Raphidascarididae; Trypanorhyncha.

Artigo Científico: Recebido em 14/03/2017; Aprovado em 30/06/2017 1Laboratório de Inspeção e Tecnologia de Pescado, Faculdade de Medicina Veterinária, Universidade Federal Fluminense, Rua Vital Brazil Filho, 64, Vital Brazil, 24320-340 Niterói, RJ, Brasil. 2Laboratório de Helmintos Parasitos de Vertebrados, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil, 4365, Manguinhos, 21040-900 Rio de Janeiro, RJ, Brasil. E-mail: [email protected] (corresponding author)

B. Inst. Pesca, São Paulo, 43(3): 385 - 398, 2017 Doi: 10.20950/1678-2305.2017v43n3p385 386 SILVA et al.

INTRODUCTION hypersensitivity has been demonstrated for some species in studies using a murine model (RODERO The tile fish, Lopholatilus villarii Miranda-Ribeiro, and CUÉLLAR, 1999; VÁZQUEZ-LÓPEZ et al., 2001, 2002; 1915 (Malacanthidae) occurs in the Southwest GÒMEZ-MORALES et al., 2008; MATTOS et al., 2013). Atlantic from Brazil to Argentina, and is known The aim of this study was to report, for the for its good tasting meat, which has commonly first time, larvae of anisakid and raphidascaridid been marketed fraudulently as mottled grouper, nematodes and trypanorhynch cestodes as parasites Mycteroperca rubra Bioch, 1793, or Argentinian of L. villarii marketed in the municipality of Niterói, sandperch, Pseudopercis semifasciata Cuvier, 1829 RJ, Brazil. Parasitological indices were calculated (FIGUEIREDO and MENEZES 1980; LIMA and and infection sites presented, with comments on MESQUITA, 1996; PAIVA and ANDRADE-TUBINO, the hygienic-sanitary significance of these parasites. 1998; FROESE and PAULY, 2016). The nematodes of the families Anisakidae and Raphidascarididae have been reported parasitizing METHODS several Brazilian marine fish in studies on classical and molecular taxonomy, ecology and hygiene and From August 2015 to September 2016, 31 health, since the larvae migrate after the death of the specimens [total length 43-71 (52.7) cm; weight hosts (FELIZARDO et al., 2009; DIAS et al., 2010,2011; 0.970 – 4.810 (1.925) kg] were acquired from fish KNOFF et al., 2012,2013; FONTENELLE et al., markets in the municipality of Niterói, state of Rio 2013,2015; RIBEIRO et al., 2014; PANTOJA et al., 2015; de Janeiro, Brazil. The fish were transported in an KURAIEM et al., 2016; FONSECA et al., 2016). Human isothermal box to the laboratory for necropsy, where infections with these nematodes have been reported they were identified according to FIGUEIREDO and in Europe (Netherlands, Spain, France, Italy, Belgium, MENEZES (1980). Denmark, England and Germany), in the Americas After necropsy, the internal organs were (United States of America, Chile, Peru and Brazil), in transferred to Petri dishes containing physiological Korea and, especially, in Japan, where more than 2,000 solution with 0.65% NaCl. The nematode larvae cases occur annually (ADAMS et al., 1997; AUDICANA found dead were fixed in AFA (70% alcohol - et al., 2002; ACHA and SZYFRES, 2003; CHAI et al., formaldehyde - acetic acid), while live larvae 2005; CRUZ et al., 2010; D’AMICO et al., 2014). were fixed in hot AFA at 60 ºC, so they would die Cestodes of the order Trypanorhyncha are of distended, preserved in a solution of 70 °GL ethanol hygienic importance because of their repugnant plus 5% glycerin and clarified with Amman’s aspect, particularly when teleostean fish present lactophenol (KNOFF and GOMES, 2012). Taxonomic massive infections in their musculature and organs classification of nematodes was in accordance with that may make commercialization infeasible PETTER and MAILLARD (1988); TIMI et al. (2001); due to sanitary inspection and/or rejected by the FELIZARDO et al. (2009); KNOFF et al. (2012) and consumer (AMATO et al., 1990; SÃO CLEMENTE FONSECA et al. (2016). et al., 2004,2007; FELIZARDO et al., 2010). This Plerocerci of trypanorhynchs were transferred order possesses a great diversity of species with to distilled water and the cysts opened under a global distributions. As adults, these worms inhabit stereomicroscope with the aid of sharp needles to the stomach and intestine of elasmobranch fish, release the larvae, which were then refrigerated for while the larval forms can infect a large number of at least 24h to permit the relaxation of scolices and teleostean fish and marine invertebrates in tropical tentacular extroversion. The larvae were then fixed in and subtropical regions, but rarely freshwater fish cold AFA, stained with Langeron´s carmine, clarified and other vertebrates (CAMPBELL and BEVERIDGE, in beechwood creosote, and preserved as whole 1994; PALM, 2004). These cestodes cause significant mounts on Canada balsam according to KNOFF and economic losses. Furthermore, accidental infections GOMES (2012). The classification of Trypanorhyncha of humans by larval trypanorhynchs have been followed PALM (2004), BEVERIDGE and JUSTINE reported due to the ingestion of raw fish meat, and (2007) and SCHAEFFNER and BEVERIDGE (2013). although they do not present zoonotic potential, Measurements were obtained by bright field recent research reports that these cestodes can cause microscopy using an Olympus BX 41 microscope. allergic disorders in humans because immunological The samples were then analyzed by bright-field

B. Inst. Pesca, São Paulo, 43(3): 385 - 398, 2017 Nematode and cestode larvae of hygienic-sanitary importance... 387 microscopy with a Zeiss Axiophot microscope Maldonado Jr, Torres, Pinto and Gomes, 2012 using Nomarski’s differential interference contrast (Figures 6a-c; 7a-c)], and one fourth instar larvae

(DIC) apparatus, and images obtained with a Canon (L4) [H. deardorffoverstreetorum (Figures 8a-c; 9a-c)]. digital camera (Power Shot A640). Some specimens Additionally, 26 fish (83.9%) were parasitized with a were prepared for scanning electron microscopy total of 414 trypanorhynch cestode plerocerci of the (SEM) as described by LOPES TORRES et al. (2013). family Otobothriidae, identified as Otobothrium sp. The samples fixed in 70% ethanol were dehydrated (Figures 10a-b; 11). in an ethanol series (70º to 100º GL), CO2 critical The morphological and morphometric data of point dried, coated in gold, and then examined and the helminths found are shown in Tables 1, 2 and 3. photographed using a SEM (Jeol JSM-6390LV), under The parasitological indices of prevalence, intensity/ 15 kV acceleration voltage. Measurements were shown mean intensity and abundance/mean abundance, as in millimeters (mm) with averages in parentheses, well as the range of infection, sites of infection and unless otherwise indicated. the CHIOC deposit numbers, are shown in Table 4. The parasitological indices of prevalence, All larval specimens of Anisakis sp., intensity, mean intensity, abundance and mean Terranova sp., Pseudoterranova sp. and various H. abundance were obtained as described by BUSH et deardorffoverstreetorum were free, and some of H. al. (1997). Representative specimens of the parasites deardorffoverstreetorum were causing granulomas in were deposited in the Helminthological Collection serosas. One Anisakis sp., Pseudoterranova sp., and of the Oswaldo Cruz Institute (CHIOC), Rio de various H. deardorffoverstreetorum were live with high Janeiro, Brazil. motility. The unique H. deardorffoverstreetorum L4 was found live and parasitizing the intestinal serosa. Only one fish possessed one H. deardorffoverstreetorum L3 RESULTS parasitizing the abdominal musculature. The trypanorhynch cestode Otobothrium sp. From the 31 specimens of L. villarii analyzed, exhibited tiny plerocerci with spherical blastocysts. 28 (90.3%) were parasitized with a total of 226 Only one fish was parasitized with a large number individual nematode larvae of Anisakidae and of cestodes (232), which were located in the intestine Raphidascarididae, 225 of which were third instar serosa where they were becoming visible and larvae (L3) [two Anisakis sp. (Figure 1 a-c), 14 Terranova producing a repugnant appearance (Figure 12). sp. (Figures 2 a-c; 3 a-c), three Pseudoterranova sp. After extroversion, most plerocerci were alive and (Figures 4 a-c; 5 a-b) and 206 Hysterothylacium exhibited moderate motility. deardorffoverstreetorum Knoff, Felizardo, Iñiguez,

Figure 1. Anisakis sp. L3 from Lopholatilus villarii observed by differential interference contrast, in lateral view. a. Anterior portion, showing larval tooth (lt), esophagus (e) and ventricle (v). b. Detail of larval tooth (lt) and excretory pore (ep). c. Posterior portion showing tail with terminal mucron (m). Bars a = 500µm; b = 50 µm; c = 100 µm.

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Figure 2. Terranova sp. L3 from Lopholatilus villarii observed by differential interference contrast, in lateral view. a. Anterior portion, showing larval tooth (lt), excretory pore (ep), esophagus (e), ventricle (v) and intestinal cecum (ic). b. Detail of larval tooth (lt) and excretory pore (ep). c. Posterior portion showing striated tail (st). Bars a = 250 µm; b = 25 µm; c = 50 µm.

Figure 3. Terranova sp. L3 from Lopholatilus villarii observed by scanning electron microscopy, in dorso-apical view. a. Anterior portion, showing larval tooth (lt). b. Posterior portion showing striated tail (st). Bars a and b = 20 µm.

Figure 4. Pseudoterranova sp. L3 from Lopholatilus villarii observed by differential interference contrast. a. Anterior portion, showing larval tooth (lt), esophagus (e), ventricle (v) and intestinal cecum (ic), in dorsal view. b. Detail of larval tooth (lt). c. Posterior portion showing tail with terminal mucron (m), in lateral view. Bars a = 200 µm; b = 50 µm; c = 100 µm.

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Figure 5. Pseudoterranova sp. L3 from Lopholatilus villarii observed by scanning electron microscopy. a. Anterior portion, showing larval tooth (lt) and excretory pore (ep), in ventro-lateral view. b. Posterior portion showing tail with terminal mucron (m), in lateral view. Bars a and b = 10 µm.

Figure 6. Hysterothylacium deardorffoverstreetorum L3 from Lopholatilus villarii observed by differential interference contrast. a. Anterior portion, showing esophagus (e), ventricle (v) and ventricular appendix (va), in dorsal view. b. Posterior portion showing tail with characteristic mucron (m), in dorsal view. c. Detail of mucron (m). Bars a = 200 µm; b = 50 µm; c = 10 µm.

Figure 7. Hysterothylacium deardorffoverstreetorum L3 from Lopholatilus villarii observed by scanning electron microscopy. a. Anterior portion, in lateral view. b. Posterior portion showing tail with characteristic mucron (m), in lateral view. c. Detail of mucron (m). Bars a and b = 20 µm; c = 5 µm.

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Figure 8. Hysterothylacium deardorffoverstreetorum L4 from Lopholatilus villarii observed by differential interference contrast. a. Anterior portion, showing esophagus (e), ventricle (v), ventricular appendix (va), and intestinal cecum (ic), in lateral view. b. Posterior portion showing characteristic multi-spinuos (ms) tail, in lateral view. c. Detail of multi-spinous tail (ms). Bars a and b = 200 µm; c = 25 µm.

Figure 9. Hysterothylacium deardorffoverstreetorum L4 from Lopholatilus villarii observed by scanning electron microscopy. a. Anterior portion, in ventro-lateral view. b. Posterior portion showing characteristic multi-spinuos (ms) tail, in ventro-lateral view. c. Detail of multi-spinous tail. Bars a and b = 20 µm; c = 5 µm.

Figure 10. Otobothrium sp. plerocercus from Lopholatilus villarii observed by differential interference contrast. a. Entire worm, showing tentacles (T), pars bothrialis (Pbo), pars vaginalis (Pv), pars bulbosa (Pb), velum (V) and appendix (Ap), lateral view. b. Detail of extroverted tentacle, indicating basal armature (BA) and metabasal armature (MA) regions, external surface. Bars a = 100; b = 20 µm.

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Figure 11. Otobothrium sp. plerocercus from Lopholatilus villarii by observed by scanning electron microscopy. Entire worm, showing tentacles (T), bothrial pits (Bp) and appendix (Ap), lateral view. Bar = 50 µm.

Table 1. Morphological and morphometric data of third instar anisakid larvae collected from Lopholatilus villarii marketed in the municipality of Niterói, state of Rio de Janeiro, Brazil.

Anisakis sp. Terranova sp. Pseudoterranova sp. (n = 1) (n = 9) (n = 1) Body (L) 23.63 6.63-10.44 (8.73) 11.5 Body (W) 0.38 0.20-0.33 (0.29) 0.25 Larval tooth 0.013 0.010-0.015 (0.012) 0.012 Excretory pore a below the larval tooth below the larval tooth below the larval tooth Nerve ring b 0.30 0.19-0.25 (0.21) 0.36 Esophagus (L) 1.75 0.92-1.30 (1.08) 1.33 Ventriculus (L) 0.70 0.31-1.35 (0.62) 0.60 Ventriculus (W) 0.20 0.10- 0.25 (0,20) 0.11 Ventricular appendix Absent Absent Absent Intestinal cecum Absent 0.33-0.60 (0.46) 0.59 Tail (L) 0.10 0.12-0.17 (0.15) 0.15 Mucron (L) 0.025 Absent 0.012 a Inconspicuous in some specimens. b Distance to the anterior end. L = length. W = width. n = number of measured specimens.

Table 2. Morphological and morphometric data of third and fourth instar larvae of Hysterothylacium deardorffoverstreetorum collected from Lopholatilus villarii marketed in the municipality of Niterói, state of Rio de Janeiro, Brazil.

Hysterothylacium Hysterothylacium

deardorffoverstreetorum L3 deardorffoverstreetorum L4 (n= 45) (n=1) Body (L) 1.63-9.25 (4.44) 10.2 Body (W) 0.08-0.25 (0.14) 0.45 Larval tooth Absent Absent Excretory porea 0.15-0.54 (0.37) - Nerve ringb 0.15-0.38 (0,27) 0.21 Esophagus (L) 0.15-0.82 (0.41) 0.93 Ventriculus (L) 0.03-0.11 (0.05) 0.09 Ventriculus (W) 0.03-0.10 (0.07) 0.13 Ventricular appendix 0.14-0.83 (0.48) 0.73 Intestinal caecum 0.03-0.18 (0.08) 0.18 Tail (L) 0.09-0.30 (0.14) 0.15 Mucron (L) 0.002-0.004 (0.003) Absent a Inconspicuous in some specimens. b Distance to the anterior end. L = Length. W = width. n = number of measured specimens.

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Table 3. Morphological and morphometric data of Otobothrium sp. plerocercus collected from Lopholatilus villarii marketed in the municipality of Niterói, state of Rio de Janeiro, Brazil. Otobothrium sp. (n=22) Scolex (L) 198-303 (249) Scolexa (W) 105-175 (144) Appendix (L ) 33-70 (54) Pbo (L) 88-195 (165) Pbo (W) 113-168 (137) Pv (L) 125-213 (16) Pb (L) 63-110 (80) Ppb Absent Velum Present Bulbs (L) 48-88 (69) Bulbs (W) 30-63 (46) Tentacle (L) 25-375 (169) Tentacle (WBI) 4-15 (11) Tentacle (WBS) 9-17 (13) Tentacle (WM) 7-12 (10) Tentacle (WA) 5-10 (9) Pbo end to pb beginningb 0-58 (22) Bulb ratioc 1 : 1.4 Scolex ratiod 2.2 : 2.4 : 1 L = Length. W = Width. Pars bothrialis = pbo. Pars vaginalis = pv. Pars bulbosa = pb. Pars post-bulbosa = ppb. aMaximum width at level of pb. bDistance. c W : L. d pbo : pv : pb. WBI = width at basal insertion. WBS = width at basal swelling. WM = width at metabasal. WA = width at apical. Measurements are presented in micrometers.

Table 4. Parasitological indices of prevalence (P), intensity/mean intensity (I/MI), abundance/ mean abundance (A/MA), range of infection (RI) and sites of infection (SI), and numbers of deposit in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC) of the anisakid and raphidascaridid nematode larvae and Otobothrium sp. plerocercus cestode from Lopholatilus villarii marketed in the municipality of Niterói, state of Rio de Janeiro, Brazil. P (%) I/MI A/MA RI SI CHIOC Nematoda Anisakis sp. 3.2 2* 0.06* - IS 38366 Terranova sp. 22.6 2 0.45 1-4 IS, SS 38364, 38365, 38367 Pseudoterranova sp. 6.4 1.5 0.09 1-2 IS 38368 Hysterothylacium IS, SS, LS, 87.1 7.7 6.68 1-28 38369, 38370 deardorffoverstreetorum AM Cestoda Otobothrium sp. 83.9 15.9 13.35 1-232 IS, SS, LS 38469, 38470, 38471a-c * Only the intensity is shown, because only one host was parasitized. IS = intestinal serosa. SS = stomach serosa. LS = liver serosa. AM = abdominal musculature.

Figure 12. Otobothrium sp. plerocerci with tiny spherical blastocysts (indicated by arrows) in large numbers attached to the intestinal serosa of Lopholatilus villarii. Bar = 2 cm.

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Remarks Cuvier, 1829, all hosts collected from the state of

The specimens of Anisakis sp. (L3) exhibited Rio de Janeiro (KNOFF et al. 2012; FONTENELLE et morphological and morphometric features similar al. 2013; RIBEIRO et al. 2014; KURAIEM et al. 2016; to those of the larval specimens of Anisakis simplex FONSECA et al. 2016). (Rudolphi, 1809) (=Anisakis sp.) reported by The majority of H. deardorffoverstreetorum were FELIZARDO et al. (2009), Anisakis type I by SAAD et collected from the interior of small granulomas, as al. (2012), Anisakis sp. by FONTENELLE et al. (2013) was also observed by FELIZARDO et al. (2009) and and KURAIEM et al. (2016) and A. typica (Diesing, KURAIEM et al. (2016).

1860) by FONSECA et al. (2016) all from teleostean The H. deardorffoverstreetorum (L4) specimen hosts collected from the state of Rio de Janeiro, Brazil. found in the present study is in agreement with the

The specimens of Terranova sp. (L3) possessed morphology and morphometry of Hysterothylacium larval tooth, conic and striated tail, absent mucron, sp. n° 2 and H. deardorffoverstreetorum (L4) reported elongated ventriculus (less than 7 times longer by PETTER and MAILLARD (1988) and FELIZARDO than wide) and intestinal cecum, all diagnostic et al. (2009), respectively. These larvae possesses characteristics of the genus. When compared to morphology distinguishable from that of L3, the specimens of other reports, one morphological including having well-developed lips, posterior end difference was observed; the length of the intestinal with a caudal multi-spinous structure and mucron cecum is approximately half the length of the absent. ventriculus, while those Terranova sp. specimens The specimens of Otobothrium sp. are in from Engraulis anchiota Hubbs & Marini, 1935, from accordance with the morphological features of Argentina and Uruguay (TIMI et al., 2001) and the genus presented in previous descriptions and Paralichthys isosceles Jordan, 1890, P. patagonicus revisions (PALM, 2004; BEVERIDGE and JUSTINE, Jordan, 1889, Xystreurys rasile (Jordan, 1891) and 2007; SCHAEFFNER and BEVERIDGE, 2013). There Cynoscion guatucupa (Cuvier, 1830) from state of Rio are 11 valid species of Otobothrium: O. alexanderi de Janeiro, Brazil, had an intestinal cecum that was Palm, 2004; O. australe Palm, 2004; O. carcharidis always almost twice the length of the ventriculus (Shipley & Hornell, 1906) Pintner, 1913; O. crenacolle (FELIZARDO et al., 2009; FONTENELLE et al., 2013; Linton, 1890; O. curtum (Linton, 1909) Dollfus, 1942; FONSECA et al., 2016). O. insigne Linton, 1905; O. minutum Subhapradha,

Pseudoterranova sp. (L3) specimens possessed a 1955; O. mugilis Hiscock, 1954; O. parvum Beveridge dorsal lip bearing a pair of double papillae, two & Justine, 2007; O. penetrans Linton, 1907; and ventrolateral lips, a tiny boring tooth between the O. propecysticum Dollfus, 1969 (BEVERIDGE and ventrolateral lips, an excretory pore at the base of JUSTINE, 2007; SCHAEFFNER and BEVERIDGE, the ventrolateral lips, a long ventriculus, absent 2013). The specimens of Otobothrium sp. are ventricular appendix, intestinal cecum and a tail morphometricaly similar to four species, O. curtum, with a mucron, which were in agreement with the O. minutum, O. parvum and O. propecysticum, in description of P. decipiens (Krabbe, 1878) (L3) collected possessing an extremely short scolex, but they from Gadus morhua L., 1758 fillets marketed in Nova differ from O. curtum in the presence of bothrial Scotia, Canada and Brazil, Thrysites atun (Euphrasen, pits, and from O. minutum by the presence of little 1791) from New Zealand and Trigla lucerna L., 1758, or no space between the end of the pars bothrialis from the Mediterranean Sea, France (MCCLELLAND, and the beginning of the pars bulbosa (PALM, 2004; 1980; HURST, 1984; PETTER and MAILLARD, 1988; BEVERIDGE and JUSTINE, 2007). MAFRA et al., 2015). Morphometrically, specimens of the present study had a body size approximately three times smaller than those reported by the DISCUSSION aforementioned authors. The parasitological indices of the anisakid and The specimens of H. deardorffoverstreetorum (L3) found in L. villarii exhibited similar morphological raphidascaridid nematodes were compared with and morphometric features to the specimens from those for other teleostean species from along the coast P. isosceles, P. patagonicus, X. rasile, C. guatucupa, of the state of Rio de Janeiro reported since 2001. Chaetodipterus faber (Broussonet, 1782), Trachinotus The specimens of Anisakis sp. had indices carolinus (Linnaeus, 1766), and Priacanthus arenatus similar to those reported for A. physeteris (Baylis,

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1923), A. simplex and Anisakis sp. from Genypterus was not found occurring with other trypanorhynch brasiliensis Regan, 1903 (KNOFF et al., 2007); A. species. simplex from P. isosceles (FELIZARDO et al., 2009) Species of Otobothrium have been reported, and and from Lophius gastrophysus Miranda-Ribeiro, 1915 are now well known, for their ability to invade and (SAAD et al., 2012); Anisakis sp. from C. guatucupa inhabit fish meat, thus causing economic losses in (FONTENELLE et al., 2013); and A. typica from P. teleostean fish with commercial importance (PALM patagonicus (FONSECA et al., 2016). Its indices were et al., 1994). PALM et al. (2000) reported a massive slightly lower than those for Anisakis type I from L. infection by O. cysticum (=Otobothrium sp.) plerocerci gastrophysus (SAAD et al., 2012); Anisakis sp. from in the musculature of Peprilus burti Fowler, 1944 and P. arenatus (KURAIEM et al., 2016); A. typica from P. alepidotus (Linnaeus, 1766) in the Gulf of Mexico, Trichuris lepturus (Linnaeus, 1758) (BORGES et al., where the species has varied in prevalence from 2012) and X. rasile (FONSECA et al., 2016). 20% to 100% annually since 1970, with a range of The indices for the specimens of Terranova sp. were infection of 1-3500 plerocerci per fish. In the present higher than those for specimens from G. brasiliensis study, this trypanorhynch cestode was not found in (KNOFF et al., 2007), P. isosceles (FELIZARDO et al., the musculature. 2009), L. gastrophysus (SAAD et al., 2012), C. guatucupa and Selene setapinis (Mitchill, 1815) (FONTENELLE et al., 2013, 2015) and from X. rasile (FONSECA et al., CONCLUSIONS 2016); and were lower than those from P. patagonicus (FONSECA et al., 2016). This is the first record of anisakid and The indices for the specimens of Pseudoterranova raphidascaridid nematodes and trypanorhynch sp. were similar to those of specimens from Caranx cestodes in L. villarii. hippos (Linnaeus, 1766) (LUQUE and ALVES, 2001), The presence of the larvae of these nematodes in G. brasiliensis (ALVES et al., 2002, KNOFF et al., 2007) organ serosas should not be underestimated, since and Tylosurus acus (Lacépède, 1803) (TAVARES et al., they are capable of migrating from the time of capture 2004), and to those of P. decipiens from G. brasiliensis to commercialization, which can take a few days. This (KNOFF et al., 2007); and were lower than those from was the case for the unique H. deardorffoverstreetorum Caranx latus Agassiz, 1831 (LUQUE and ALVES, larva found in the abdominal musculature in the 2001). present study, which would be sufficient to cause an The indices for the specimens of H. accidental human infection, through the ingestion of deardorffoverstreetorum were much higher than those improperly processed meat of this fish. for Hysterothylacium sp. from G. brasiliensis (KNOFF In addition, antigens from these nematodes can be et al., 2007). Additionally, the range of infection released into fish meat and cause allergic disorders. It was more than one and half to four times higher is suggested that additional research be undertaken than those of Hysterothylacium sp. from Trachinotus to evaluate the knowledge used for diagnosis of not carolinus (Linnaeus, 1766) and Chaetodipterus faber only nematodes but also trypanorhynch cestodes. (Broussonet, 1782) (RIBEIRO et al., 2014) and T. This research should address the potential risk of lepturus (BORGES et al., 2012); and as high as those H. these helminths to humans and potential for control deardorffovestreetorum from P. isosceles (FELIZARDO and prevention of the parasitic diseases they cause. et al., 2009), C. guatucupa (FONTENELLE et al., 2013), Although trypanorhynch cestodes have not been P. arenatus (KURAIEM et al., 2016), P. patagonicus and found in musculature, their massive presence in the X. rasile (FONSECA et al., 2016). intestinal serosa of the one fish collected produced In Brazil, specimens of Otobothrium sp. have been a repugnant appearance, which leads to loss of the reported from various teleostean fish, including commercial value or complete rejection of the fish by Balistes vetula (Linnaeus, 1758) (SÃO CLEMENTE the consumer. Thus, it is suggested that fish viscera et al., 1995; ALVES et al., 2005); G. brasiliensis (SÃO be removed when marketed. CLEMENTE et al., 2004); and P. isosceles (FELIZARDO et al., 2010); and as O. cysticum (=Otobothrium sp.) in mesentery of Scomberomorus maculatus (Mitchill, ACKNOWLEDGMENTS 1815) and Sphyraena guachancho (Cuvier, 1829) (PALM, 1997). In the present study, Otobothrium sp. The authors would like to thank Ricardo Baptista

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Schmidt (Serviço de Produção e Tratamento de Malaysia, with the description of O. parvum n. Imagens do Instituto Oswaldo Cruz, FIOCRUZ) for sp. Zootaxa, 1587: 1–25. processing the figures; the Rudolf Barth Electron Microscopy Platform of the Oswaldo Cruz Institute BORGES, J.; CUNHA, L.; SANTOS, H.; MONTEIRO- / Fiocruz for producing the SEM images; and the NETO, C.; SANTOS, C. 2012 Morphological Conselho Nacional de Desenvolvimento Científico and molecular diagnosis of anisakid nematode e Tecnológico (CNPq) for financial support. larvae from cutlassfish Trichiurus( lepturus) off the coast of Rio de Janeiro, Brazil. PLoS ONE, 7(7): e40447. REFERENCES BUSH, A.O.; LAFFERTY, K.D.; LOTZ, J.M.; ACHA, P.N.; SZYFRES, B. 2003 Zoonoses and SHOSTAK, A.W. 1997 Parasitology meets communicable diseases common to man and animals. ecology on its own terms: Margolis et al. 3º ed. Washington, D.C.: PAHO. 395 p. revisited. Journal of Parasitology, 83(4): 575-583.

ADAMS, A.M.; MURREL, K.D.; CROSS, J.H. 1997 CAMPBELL, R.A.; BEVERIDGE, I. 1994 Order Parasites of fish and risk to public health. Trypanorhyncha Diesing, 1863. In: KHALIL, Scientifical and Technical Review of the Office L.F.; JONES, A.; BRAY, R.A. (Eds.). Keys to the International des Epizooties, 16(2): 652-660. cestodes parasites of vertebrates. Cambridge: CABI. p.51148. ALVES, D.R.; LUQUE, J.L.; PARAGUASSÚ, A.R. 2002 Community ecology of the metazoan CHAI, J.Y.; MURRELL, K.D.; LYMBERY, A.J. 2005 parasites of pink cusk-eel, Genypterus brasiliensis Fish-borne parasitic zoonoses: status and issues. (Osteichthyes: Ophidiidae), from the coastal International Journal for Parasitology, 35(11-12): zone of the state of Rio de Janeiro. Memórias do 1233-1254. Instituto Oswaldo Cruz, 97(5): 683-689. CRUZ, A.R.; SOUTO, P.C.S.; FERRARI, C.K.B.; ALVES, D.R.; PARAGUASSÚ, A.R.; LUQUE, J.L. ALLEGRETTI, S.M.; ARRAIS-SILVA, W.W. 2010 2005 Community ecology of the metazoan Endoscopic imaging of the first clinical case of parasites of the grey triggerfish,Balistes capriscus Anisakidosis in Brazil. Scientia Parasitologica, Gmelin, 1789 and queen triggerfish B. vetula 11(2): 97-100. Linnaeus, 1758 (Osteichthyes: Balistidae) from the State of Rio de Janeiro, Brazil. Revista D’AMICO, P.; MALANDRA, R.; COSTANZO, F.; Brasileira de Parasitologia Veterinária, 14(2): 71-77. CASTIGLIEGO, L.; GUIDI, A.; GIANFALDONI, D.; ARMANI., A. 2014 Evolution of the Anisakis AMATO, J.F.R.; SÃO CLEMENTE, S.C.; OLIVEIRA, risk management in the European and Italian G.A. 1990 Tentacularia coryphaenae Bosc, 1801 contexto. Food Research International, 64(1): 348-362. (Eucestoda: Trypanorhyncha) in the inspection and technology of the Skipjack Tuna, Katsuwonus DIAS, F.J.E.; SÃO CLEMENTE, S.C.; KNOFF, M. pelamis (L.) (Pisces: Scombridae). Atlântica, 12(1): 2010 Nematoides anisaquídeos e cestoides 73-77. Trypanorhyncha de importância em saúde pública em Aluterus monoceros (Linnaeus, 1758) AUDICANA, M.T.; ANSOTEGUI, I.J.; CORRES, no Estado do Rio de Janeiro, Brasil. Revista L.F.; KENEDDY, M.W. 2002 Anisakis simplex: Brasileira de Parasitologia Veterinária, 19(2); 94-97. dangerous – dead and alive? Trends in Parasitology, 18(1): 20-25. DIAS, F.J.E.; SÃO CLEMENTE, S.C.; PINTO, R.M.; KNOFF, M. 2011 Anisakidae nematodes and BEVERIDGE, I.; JUSTINE, J.L. 2007 Redescriptions Trypanorhyncha cestodes of hygienic importance of four species of Otobothrium Linton, 1890 infecting the king mackerel Scomberomorus (Cestoda: Trypanorhyncha), including new cavalla (Osteichthyes: Scombridae) in Brazil. records from Australia, New Caledonia and Veterinary Parasitology, 175(3-4): 351-355.

B. Inst. Pesca, São Paulo, 43(3): 385 - 398, 2017 396 SILVA et al.

FELIZARDO, N.N; KNOFF, M.; PINTO, R.M; HURST, R.J. 1984 Identification and description GOMES, D.C. 2009 Larval anisakid nematodes of larval Anisakis simplex and Pseudoterranova of the flounder Paralichthys isosceles Jordan, decipiens (Anisakidae: Nematoda) from New 1890 (Pisces: Teleostei) from Brazil. Neotropical Zealand waters. New Zealand Journal of Marine Helminthology, 3(2): 57-64. and Freshwater Research, 18(2): 177-186.

FELIZARDO, N.N.; TORRES, E.J.L.; FONSECA, KNOFF, M.; GOMES, D.C. 2012 Capítulo 3. M.C.G.; PINTO, R.M.; GOMES, C.D.; Metodologia básica para coleta e processamento KNOFF, M. 2010 Cestodes of the flounder de helmintos parasitos. In: MOLINARO, E.; Paralichthys isosceles Jordan, 1890 (Osteichthyes - CAPUTO, L.; AMENDOEIRA, R. Conceitos e Paralichthyidae) from the State of Rio de Janeiro, Métodos para formação de profissionais em laboratórios Brazil. Neotropical Helminthology, 4(2): 113-125. de saúde: volume 5. Ed. Escola Politécnica de Saúde Joaquim Venâncio, Fundação Oswaldo Cruz, Rio FIGUEIREDO, J.L.; MENEZES, N.A. 1980 Manual de de Janeiro. p. 251-281. peixes marinhos do Sudeste do Brasil. III. Teleostei (2). São Paulo: Universidade de São Paulo, KNOFF, M.; FELIZARDO, N.N.; IÑIGUEZ, A.M.; Museu de Zoologia. 93 p. MALDONADO, A.J.R.; TORRES, E.J.L.; PINTO, R.M.; GOMES, D.C. 2012 Genetic and FONSECA, M.C.G.; KNOFF, M.; FELIZARDO, morphological characterisation of a new species N.N.; DI AZEVEDO, M.I.N.; LOPES TORRES, of the genus Hysterothylacium (Nematoda) E.J.; GOMES, D.C.; IÑIGUEZ, A.M.; SÃO from Paralichthys isosceles Jordan, 1890 (Pisces: CLEMENTE, S.C. 2016 Integrative taxonomy of Teleostei) of the Neotropical region, state of Rio Anisakidae and Raphidascarididae (Nematoda) de Janeiro, Brazil. Memórias do Instituto Oswaldo in Paralichthys patagonicus and Xystreurys rasile Cruz, 107(2): 186-193. (Pisces: Teleostei) from Brazil. International Journal of Food Microbiology, 235(1): 113–124. KNOFF, M.; SÃO CLEMENTE, S.C.; FONSECA, M.C.G.; ANDRADA, C.G.; PADOVANI, R.E.S.; FONTENELLE, G.; KNOFF, M.; FELIZARDO, GOMES, D.C. 2007 Anisakidae parasitos de N.N.; LOPES, L.M.S.; SÃO CLEMENTE, S.C. 2013 congro-rosa, Genypterus brasiliensis Regan, 1903 Nematodes of zoonotic importance in Cynoscion comercializados no estado do Rio de Janeiro, guatucupa (Pisces) in the state of Rio de Janeiro. Revista Brasil de interesse na saúde pública. Parasitologia Brasileira de Parasitologia Veterinária, 22(2): 281-284. Latinoamericana, 62(3-4): 127-133.

FONTENELLE, G.; KNOFF, M.; FELIZARDO, N.N.; KNOFF, M.; SÃO CLEMENTE, S.C.; FONSECA, LOPES TORRES, E.J.; LOPES, L.M.S.; GOMES, M.C.G.; FELIZARDO, N.N.; LIMA, F.C.; PINTO, D.C.; SÃO CLEMENTE, S.C. 2015 Anisakidae R.M.; GOMES, D.C. 2013. Anisakidae nematodes and Raphidascarididae larvae parasitizing Selene in the blackfin goosefish, Lophius gastrophysus setapinnis (Mitchill, 1815) in the State of Rio de Miranda-Ribeiro, 1915 purchased in the State of Janeiro, Brazil. Brazilian Journal of Veterinarian Rio de Janeiro, Brazil. Acta Scientiarum. Biological Parasitology, 24(1): 72-77. Sciences, 35(1): 129-133.

FROESE, R.; PAULY, D. 2016 FishBase. World Wide KURAIEM, B.P.; KNOFF, M.; FELIZARDO, N.N.; Web electronic publication. Available from:< GOMES, D.C.; SÃO CLEMENTE, S.C. 2016 http://www.fishbase.org/summary/15383> Nematode larvae infecting Priacanthus arenatus Access in: Nov. 28 2016. Cuvier, 1829 (Pisces: Teleostei) in Brazil. Anais da Academia Brasileira de Ciências, 88(2): 857-863. GÒMEZ-MORALES, M.A; LUDOVISI, A.; GIUFFRA, E.; MANFREDI, M.T.; PICCOLO, G.; POZIO, E. 2008 LIMA, F.C; MESQUITA, E.F.M. 1996 Fraudes detectadas Allergenic activity of Molicola horridus (Cestoda, na comercialização de pescado no município de Trypanorhyncha), a cosmopolitan fish parasite, in a Niterói, Estado do Rio de Janeiro, Brasil. Revista mouse model. Veterinary Parasitology, 157(3-4): 314-320. Brasileira de Ciências Veterinária, 3(2): 39-43.

B. Inst. Pesca, São Paulo, 43(3): 385 - 398, 2017 Nematode and cestode larvae of hygienic-sanitary importance... 397

LOPES TORRES, E.J; SOUZA, W. MIRANDA, K. Trypanorhynchid cestodes of commercial 2013 Comparative analysis of Trichuris muris inshore fishes of the West African coast.Aquatic surface using conventional, low vacuum, Living Resources, 7(3): 153-164. environmental and field emission scanning electron microscopy. Veterinary Parasitology, PANTOJA, C.S.; BORGES, J.N.; SANTOS, C.P.; 196(3-4): 409-416. LUQUE, J.L. 2015 Molecular and morphological characterization of anisakid nematode larvae LUQUE, J.L.; ALVES, D.R. 2001 Ecologia das from the sandperches Pseudopercis numida comunidades de metazoários parasitos, do and Pinguipes brasilianus (Perciformes: xaréu, Caranx hippos (Linnaeus) e do xerelete, Pinguipedidae) off Brazil. Journal of Parasitology, Caranx tatus Agassiz (Osteichthyes, Carangidae) 101(4): 492–499. do litoral do estado do Rio de Janeiro, Brasil. Revista Brasileira de Zoologia, 18(2): 399 - 410. PETTER, J.A.; MAILLARD, C. 1988 Larves d’ascarides parasites de poissons em Méditerranée MAFRA, C.; MANTOVANI, C.; BORGES, J.N.; Occidentale. The Bulletin of the Natural History BARCELOS, R. M.; SANTOS, C.P. 2015 Museum, 10(2): 347-369. Morphological and molecular diagnosis of Pseudoterranova decipiens (sensu stricto) RIBEIRO, J.; SÃO CLEMENTE, S.C.; LOPES, L.M.S.; (Anisakidae) in imported cod sold in KNOFF, M. 2014 Nematode larvae of hygienic Brazil. Brazilian Journal of Veterinary Parasitology, importance infecting Chaetodipterus faber 24(2): 209-215. (Broussonet, 1782) and Trachinotus carolinus (Linnaeus, 1766) (Pisces: Teleostei) in Brazil. MATTOS, D.P.B.G; VERÍCIMO, M.A.; SÃO Revista Brasileira de Medicina Veterinária, 36(2): CLEMENTE, S.C. 2013 O Pescado e os Cestoides 121-124. Trypanorhyncha – Do Aspecto Higiênico ao potencial alergênico. Veterinária Notícias, 19(2): 127-139. RODERO, M.; CUÉLLAR, C. 1999 Humoral immune responses induced by Gymnorhynchus gigas MCCLELLAND, G. 1980 Phocanema decipiens. extracts in BALB/c mice. Journal of Helminthology, Molting in seals. Experimental Parasitology, 49(1): 73(3): 239-243. 128-136. SAAD, C.D.R.; VIEIRA, F.M.; LUQUE, J.L. 2012 PAIVA, M.P.; ANDRADE-TUBINO, M.F. 1998 Larvae of Anisakidae Skrjabin & Karokhin, 1945 Distribuição e abundância de peixes bentônicos (Nematoda, Ascaridoidea) in Lophius gastrophysus explotados pelos linheiros ao largo do sudeste Miranda-Ribeiro, 1915 (Actinopterygii, do Brasil (1986-1995). Revista Brasileira de Lophiidae) from the coastal zone of the state of Biologia, 58(4): 619-632. Rio de Janeiro, Brazil. Neotropical Helminthology, 6(2): 159-177. PALM, H.W. 1997 Trypanorhynch cestodes of commercial fishes from northeast Brazilian SÃO CLEMENTE, S.C.; KNOFF, M.; PADOVANI, coastal waters. Memórias do Instituto Oswaldo R.E.S.; LIMA, F.C.; GOMES, D.C. 2004 Cruz, 92(1): 69–79. Cestóides Trypanhorhyncha parasitos de congro-rosa, Genypterus brasiliensis, Regan,1903 PALM, H.W. 2004 The Trypanorhyncha Diesing, 1863. comercializados nos municípios de Niterói Bogor: PKSPL-IPB Press. 710 p. e rio de Janeiro, Brasil. Revista Brasileira de Parasitologia Veterinária, 13(3): 97-102. PALM, H.W; MUNDT, U.; OVERSTREET, R. 2000 Sensory receptors and surface ultrastructure SÃO CLEMENTE, S.C.; KNOFF, M.; LIMA, F.C.; within trypanorhynch cestodes. Parasitology ANDRADA, C.D.G.; FELIZARDO, N.N.; Research, 86(10): 821–833. PADOVANI, R.E.S.; GOMES, D.C. 2007 Cestóides Trypanorhyncha parasitos de peixe PALM, H.; OBIEKEZIE, A.; MÖLLER, H. 1994 sapo-pescador, Lophius gastrophysus Miranda-

B. Inst. Pesca, São Paulo, 43(3): 385 - 398, 2017 398 SILVA et al.

Ribeiro, 1915 comercializados no Estado do Rio de Janeiro, Brasil. Revista Brasileira de Parasitologia Veterinária, 16(1): 37-42.

SÃO CLEMENTE, S.C; LIMA, F.C; UCHOA, C.M. 1995 Parasitos de Balistes vetula (L.) e sua importância na inspeção do pescado. Revista Brasileira de Ciência Veterinária, 2(2): 39-41.

SCHAEFFNER, B.C.; BEVERIDGE, I. 2013 Redescriptions and new records of species of Otobothrium Linton, 1890 (Cestoda: Trypanorhyncha). Systematic Parasitology, 84(1): 17-55.

TAVARES, L.E.R.; BICUDO, A.J.A.; LUQUE, J.L. 2004 Metazoan parasites of needlefishTylosurus acus (Lacépède, 1803) (Osteichthyes: Belonidae) from the coastal zone of the state of Rio de Janeiro, Brazil. Revista Brasileira de Parasitologia Veterinaria, 13(1): 36-40.

TIMI, J.T; SARDELLA, N.H; NAVONE, G.T. 2001 Parasitic nematodes of Engraulis anchoita Hubbs et Marini, 1935 (Pisces, engraulidae) of the Argentine and Uruguayan coasts, South West Atlantic. Acta Parasitologica, 46(3): 186-193.

VÁZQUEZ-LÓPEZ, C.; ARMAS-SERRA, C.; BERNARDINA, W.; RODRÍGUEZ-CAABEIRO, F. 2001 Oral inoculation with Gymnorhynchus gigas induces anti-parasite anaphylatic antibody production in both mice and rats and adverse reactions in challenge mice. International Journal of Food Microbiology, 64(3): 307-315.

VÁZQUEZ-LÓPEZ, C.; ARMAS-SERRA, C.; BERNARDINA, W.; RODRÍGUEZ-CAABEIRO, F. 2002 A 24-kDa collagenase from Gymnorhynchus gigas elicits rat ileum hyperreactivity and is a target of humoral responses in mice previously given a single oral dose of parasite extract. Digestive Diseases and Sciences, 47(4): 935-942.

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